Three RNA Polymerase II Carboxyl-terminal Domain Kinases Display Distinct Substrate Preferences

Abstract

CDK7, CDK8, and CDK9 are cyclin-dependent kinases (CDKs) that phosphorylate the C-terminal domain (CTD) of RNA polymerase II. They have distinct functions in transcription. Because the three CDKs target only serine 5 in the heptad repeat of model CTD substrates containing various numbers of repeats, we tested the hypothesis that the kinases differ in their ability to phosphorylate CTD heptad arrays. Our data show that the kinases display different preferences for phosphorylating individual heptads in a synthetic CTD substrate containing three heptamer repeats and specific regions of the CTD in glutathione S-transferase fusion proteins. They also exhibit differences in their ability to phosphorylate a synthetic CTD peptide that contains Ser-2-PO 4 . This phosphorylated peptide is a poor substrate for CDK9 complexes. CDK8 and CDK9 complexes, bound to viral activators E1A and Tat, respectively, target only serine 5 for phosphorylation in the CTD peptides, and binding to the viral activators does not change the substrate preference of these kinases. These results imply that the display of different CTD heptads during transcription, as well as their phosphorylation state, can affect their phosphorylation by the different transcription-associated CDKs.The three cyclin-dependent kinases, CDK7, CDK8, and CDK9, 1 have an established connection with transcription machinery and are regulated by constitutively expressed cyclins. These CDKs can hyperphosphorylate the CTD of the large subunit of RNA polymerase II (pol II) and therefore are CTD kinases (1). The CTD of mammalian pol II consists of 52 heptad repeats with the consensus sequence 1 YSPTSPS 7 , which is especially well-conserved in the first half (N-terminal) of the CTD. Of the phosphorylatable amino acid residues, serine 5 is the most conserved, with only one threonine replacement, indicating that it is likely to have a crucial role in the function of the CTD. Pol II CTD is highly phosphorylated in vivo, mostly at serine (positions 2 and 5) but also at threonine and tyrosine (2). Hyperphosphorylated CTD is correlated with transcription initiation and elongation, whereas the holoenzyme and preinitiation complexes contain pol II with hypophosphorylated CTD (3). In yeast cells, replacing either serine 2 or serine 5 with alanine in each of this organism's 26 CTD heptad repeats causes lethality (4).The functions of these kinases correlate with different stages of transcription. CDK7 is a subunit of the general transcription factor TFIIH and is the kinase that phosphorylates pol II CTD after the preinitiation complex is formed (5). CDK7 is the catalytic member of a three-subunit complex called CAK (CDKactivating kinase) composed of CDK7/cyclin H and MAT-1 (6, 7). CAK is also a subassembly of TFIIH (8). Comparison between the kinase activity of CAK and TFIIH revealed differences in substrate specificity: CAK shows a strong preference for CDK2, consistent with its function in the cell cycle, but cannot hyperphosphorylate pol II; TFIIH hyperphosphorylates pol II, but CDK2 i...

Regulation of RNA polymerase II activity by CTD phosphorylation and cell cycle control

“…Results of kinetic studies with P-TEFb and puri®ed RNAP II as a substrate indicated slow initial phosphorylation events followed by more rapid phosphorylation of the CTD, suggesting that prior phosphorylation events can increase P-TEFb activity (Marshall et al, 1996). In apparent contrast, a recent study reported inhibition of P-TEFb kinase activity towards synthetic CTD peptides containing phosphoserine 2 (Ramanathan et al, 2001). Taken together, these in vitro observations strongly suggest that the activity of CTD kinases towards particular target residues in the natural RNAP II CTD substrate in vivo will depend on amino acid sequence and on the phosphorylation status of the corresponding CTD repeat.…”

“…However, it has to be kept in mind that the natural RNAP II CTD substrate is very complex and that the activity of individual CTD kinases towards speci®c CTD amino acid residues might be modulated by a number of factors. Tyrosine phosphorylation by c-Abl kinase appears to require divergent amino acid sequences at the C-terminus of the CTD (Baskaran et al, 1999), whereas CDK8 and CDK9 kinase complexes appear to preferentially phosphorylate the highly conserved Nterminal portion of the CTD (Ramanathan et al, 2001). CDK7 has been reported to phosphorylate different CTD regions of more than twenty repeats with comparable activity (Ramanathan et al, 2001).…”

“…Tyrosine phosphorylation by c-Abl kinase appears to require divergent amino acid sequences at the C-terminus of the CTD (Baskaran et al, 1999), whereas CDK8 and CDK9 kinase complexes appear to preferentially phosphorylate the highly conserved Nterminal portion of the CTD (Ramanathan et al, 2001). CDK7 has been reported to phosphorylate different CTD regions of more than twenty repeats with comparable activity (Ramanathan et al, 2001). However, preferential phosphorylation of heptapeptides with a lysine residue at position 7 over consensus heptapeptide sequences has been observed with synthetic CTD peptide substrates containing only four repeats (Rickert et al, 1999).…”

“…However, preferential phosphorylation of heptapeptides with a lysine residue at position 7 over consensus heptapeptide sequences has been observed with synthetic CTD peptide substrates containing only four repeats (Rickert et al, 1999). In addition, while CDK7 CTD kinase activity is generally thought to be speci®c for Ser-5 (Sun et al, 1998;Trigon et al, 1998;Ramanathan et al, 2001), there are indications that Ser-2 phosphorylation by CDK7 might occur within less conserved heptapeptide repeats located at the C-terminal end of the CTD (Dubois et al, 1997;Bensaude et al, 1999). Results of kinetic studies with P-TEFb and puri®ed RNAP II as a substrate indicated slow initial phosphorylation events followed by more rapid phosphorylation of the CTD, suggesting that prior phosphorylation events can increase P-TEFb activity (Marshall et al, 1996).…”

From Roscovitine to CYC202 to Seliciclib – from bench to bedside: discovery and development

“…Similar to CDK7 -CDK8 can also phosphorylate the C-terminal domain of RNA Polymerase II in vitro;however, its specificity is for residues Ser-2 and Ser-5, as opposed to the Ser-5 and Ser-7 targeted by CDK7. The cyclin partner for CDK8 is Cyclin C, which does not show cell cycle-dependent fluctuations in concentration; control of CDK8 activity is modulated rather by other factors, such as association with Mediator Complex Subunit 12 (MED12) [34][35][36][37][38]. CDK8 is part of the large multi-subunit transcriptional regulatory complex Mediator that functions in eukaryotes as a transcriptional co-activator.…”